Despite the importance of Cu/Zn Superoxide Dismutase (SOD1) in defending against oxygen radicals, we find that > 95% of SOD1 is dispensable for protection against oxidative stress. Since SODs are the only enzymes that catalyze the dismutation of O2- into H2O2, a well-established signaling molecule, we propose that SOD1 plays a pivotal role in mediating H2O2-based redox signaling networks. Indeed, our groundbreaking studies in the model eukaryote, Saccharomyces cerevisiae, identified SOD1 as a redox regulator of a signaling circuit that integrates nutrient availability to the control of enrgy metabolism (Reddi and Culotta, Cell 2013). In this pathway, glucose and O2 availability stimulates the SOD1-dependent production of H2O2, which stabilizes a highly conserved SOD1-interacting casein kinase, YCK1 that signals respiration repression. Since our preliminary data suggests that SOD1 regulates the stability of a number of substrates of the HECT E3 ubiquitin ligase, RSP5, we propose that SOD1-derived H2O2 redox regulates the activity of RSP5 or related adapter protein(s) via cysteine thiol oxidation, which in turn mediates the stability of YCK1 and other RSP5 substrates. Additionally, since our preliminary data indicate that a relatively large fraction of the thiol proteome is oxidized to sulfenic acid in a SOD1-dependent manner, we propose that SOD1 redox regulates a multitude of cellular pathways. The current proposal seeks to address the role of SOD1 in redox signaling by investigating the mechanism of redox regulation of YCK1 stability and identifying the redox targets of SOD1 on a global proteome-wide level. Towards this end, we propose three aims utilizing molecular genetics, biochemical, and mass spectrometry-based proteomic approaches in Saccharomyces cerevisiae. In Aim 1, in order to characterize the mechanisms underlying SOD1-dependent H2O2 regulation of YCK1, we will: a) undertake a genetic screen to identify factors regulating SOD1-YCK1 signaling and YCK1 stability, b) test the role of the ubiquitination pathway and, in particular, RSP5, and c) employ mass spectrometry to identify SOD1- mediated post-translational modifications (PTMs) on YCK1, and d) YCK1-interacting proteins that regulate SOD1-YCK1 signaling. In Aim 2, in order to determine the factors responsible for the interaction between SOD1 and YCK1, we will: a) ascertain if they interact directly or indirectly by determining if SOD1 and YCK1 associate in vitro by calorimetry, and b) identify residues on SOD1 and YCK1 that are required for their interaction by mutagenesis. In Aim 3, in order to determine new SOD1-dependent redox signaling networks, we will employ mass-spectrometry based redox proteomics approaches to identify protein thiols that are oxidized in a SOD1-dependent manner on a proteome wide level. Elucidation of global SOD1-regulated cellular pathways and the mechanism of SOD1 mediated YCK1 stability will uncover new aspects of H2O2- based signal transduction and redox regulation of protein stability that will have major implications for human health and disease, including the pathogenesis of various cancers and neurodegenerative disorders.